In the present study, magnetite nanoparticles (Fe3O4 NPs) incorporated polyacrylonitrile nanofibers (PANnFs) having diameter of 350-500 nm, were electrospun and directly collected onto indium tin oxide (ITO) coated glass substrate. X-ray diffraction pattern of Fe3O4-PANnFs confirmed the existence of Fe3O4 NPs within the PANnFs. Nafion was used to make Fe3O4-PANnFs more adhesive to the ITO surface. Partial hydrolyzation of Fe3O4-PANnFs/ITO electrode was done using NaOH solution for the partial conversion of nitrite group (C N) into carboxyl and amine groups that was confirmed by Fourier transform infrared spectroscopy study. The hydrolyzed Fe3O4-PANnFs/ITO electrode was used as an immobilization matrix for monoclonal antibody specific to Vitamin-D-3 (Anti-VD) via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-Hydroxysuccinimide chemistry. Bovine serum albumin (BSA) was used as a blocking agent to block the non-specific sites onto Anti-VD/Fe3O4-PANnFs/ITO electrode surface. Fabricated BSA/Anti-VD/Fe3O4-PANnFs/ITO immunoelectrode showed improved biosensing parameters for Vitamin-D-3 detection such as sensitivity of 0.90 mu A ng(-1) mL cm(-2), limit of detection of 0.12 ng mL(-1) and detection range of 10-100 ng mL(-1). The association and dissociation constants were obtained as 74.62 ng mL(-1), 4.6 ng mL(-1), respectively.